Acute Leukemia Virus Research Articles

Acute avian leukemia viruses as tools to study hematopoietic cell differentiation.

Hematopoiesis is a multistep process during which an uncommitted, self-renewing progenitor divides and differentiates along one of eight distinct lineages, each with a unique function and each expressing a unique complement of proteins enabling them to carry out these functions. In adult vertebrates, multipotent hematopoietic precursors reside within the microenvironment of the bone marrow where they undergo a progressive maturation and commitment to one of several lymphoid and nonlymphoid cell lineages. Regulation of normal hematopoietic development requires an intricate set of cues from stromal cells involving signaling via soluble growth factors, cell-cell and cell-extracellular matrix interactions (Quesenberry 1992). Analysis of the cellular intermediates in these differentiation pathways has been greatly facilitated by the development of in vitro colony forming assays for multipotent and monopotent hematopoietic precursors and the identification of cytokines which regulate their growth and differentiation (Metcale 1984, 1988). Nevertheless, analysis of the molecular mechanisms governing differentiation and commitment to a given lineage has been technically difficult as bone marrow precursors are present in relatively low frequency and reagents allowing their isolation have only recently been developed. In addition, obtaining clonal precursor cells in sufficient numbers for biochemical analysis has been problematic since these precursors have a limited life span in vitro.

Oligodeoxynucleotides antisense to the proto-oncogene c-mpl specifically inhibit in vitro megakaryocytopoiesis

The proto-oncogene c-mpl encodes a protein whose sequence shares striking homologies with members of the highly conserved hematopoietin receptor superfamily. This gene had been transduced in a truncated form by the acute leukemogenic murine Myeloproliferative leukemia virus, which exhibits the unique property of inducing factor-independent proliferation and terminal differentiation of a broad spectrum of hematopoietic progenitors. Presently, the ligand and the role of c-mpl in the regulation of normal hematopoiesis are unknown. To show the function of c-mpl, its expression was first examined in human purified hematopoietic cell populations and, then, an antisense strategy was used. By RNA-based polymerase chain reaction, c-mpl transcripts were detected in purified CD34+ cells, megakaryocytes, and platelets. Synthetic unmodified antisense oligodeoxynucleotides were derived from different regions of the c-mpl extracellular domain. On in vitro exposure of CD34+ cells, two antisense oligomers led to a 50% to 70% inhibition of c-mpl mRNA synthesis, whereas their respective sense had no effect. Furthermore, the decrease in c-mpl mRNA correlated with a significant inhibition (range, 54% to 81%) of in vitro megakaryocytic colony formation (CFU-MK), whereas the growth of erythroid (BFU-E) or granulomacrophage (CFU-GM) colonies was unaffected. The data provide first evidences that c-mpl is involved in megakaryocytopoiesis. In addition, the results raise the possibility that this proto-oncogene encodes the receptor for a new cytokine specifically regulating thrombocytopoiesis.

Oligodeoxynucleotides Antisense to the Proto-oncogene c-mpl Specifically Inhibit In Vitro Megakaryocytopoiesis

AbstractThe proto-oncogene c-mpl encodes a protein whose sequence shares striking homologies with members of the highly conserved hematopoietin receptor super-family. This gene had been transduced in a truncated form by the acute leukemogenic murine Myeloproliferative leukemia virus, which exhibits the unique property of inducing factor-independent proliferation and terminal differentiation of a broad spectrum of hematopoietic progenitors. Presently, the ligand and the role of c-mpl in the regulation of normal hematopoiesis are unknown. To show the function of c-mpl. its expression was first examined in human purified hematopoietic cell populations and, then, an antisense strategy was used. By RNA-based polymerase chain reaction, c-mpl transcripts were detected in purified CD34+ cells, megakaryocytes, and platelets. Synthetic unmodified antisense oligodeoxynucleotides were derived from different regions of the c-mpl extracellular domain. On in vitro exposure of CD34+ cells, two antisense oligomers led to a 50% to 70% inhibition of c-mpl mRNA synthesis, whereas their respective sense had no effect. Furthermore, the decrease in c-mpl mRNA correlated with a significant inhibition (range, 54% to 81 %) of in vitro megakaryocytic colony formation (CFU-MK), whereas the growth of erythroid (BFU-E) or granulomacrophage (CFU-GM) colonies was unaffected. The data provide first evidences that c-mpl is involved in megakaryocytopoiesis. In addition, the results raise the possibility that this proto-oncogene encodes the receptor for a new cytokine specifically regulating thrombocytopoiesis.

Oncogenic conversion of Ets affects redox regulation in-vivo and in-vitro.

The avian acute leukemia virus E26 encodes a fusion protein between viral Gag and the cellular transcription factors cMyb and cEts1(p68). vEts on its own transforms more mature erythroid cells. We have compared the properties of vEts and cEts1(p68). vEts interacts preferentially with an antibody that recognizes the active conformation of the DNA-binding domain. The DNA-binding activity of vEts is particularly sensitive to incubation conditions for band-shift assays, phosphorylation and modification by sulphydryl-specific reagents. Increased sensitivity is due to loss of a protective function of cEts1 C-terminal sequences. cEts2 has a related C-terminal sequence with a similar role. These results suggest that the vEts DNA-binding domain is more accessible to protein-protein interactions and to regulatory mechanisms. Indeed, vEts DNA binding is preferentially inactivated by oxidizing conditions in-vivo. We suggest that the 'open' conformation of the vEts DNA-binding domain favours interactions with other proteins or DNA and facilitates transformation.

Expression patterns of c-myb and of v-myb induced myeloid-1 (mim-1) gene during the development of the chick embryo.

The v-myb oncogene of the acute avian leukemia virus E26 encodes a transcription factor that directly regulates the promyelocyte-specific mim-1 gene (Ness, S.A., Marknell, A. and Graf, T. Cell, 59, 1115-1125). We have investigated the relationship between the c-myb proto-oncogene and the transcription of the mim-1 gene both in vitro and in vivo. We demonstrate that the c-myb protein can transactivate the transcription of mim-1 in a transient transfection assay. In the chick embryo, we confirm that mim-1 is specifically expressed during granulopoiesis and we show that the expression of c-myb and mim-1 are perfectly correlated in the granulocytic spleen and pancreas. However we suggest that mim-1 is efficiently transcribed in the absence of c-myb in the yolk sac and in the promyelocytes at the onset of the colonization of the bursa of Fabricius. On the other hand c-myb transcripts detected in the early hemopoietic progenitor cells, in lymphoid cells and in proliferative epithelia are never associated with mim-1 transcription. We conclude that the granulocyte-specific mim-1 gene is regulated by c-myb-dependent and c-myb-independent mechanisms depending upon the environment in which granulocytic precursor cells differentiate.

V-myb and v-ets transform chicken erythroid cells and cooperate both in trans and in cis to induce distinct differentiation phenotypes.

E26 is an acute avian leukemia virus that encodes the transcriptional activator oncogenes v-myb and v-ets in a single fusion protein. This virus is also unique in that it is able to transform hematopoietic cells of both the myeloid and the erythroid lineage. To determine the contributions of v-myb and v-ets to the transforming potential of the virus, derivatives expressing separate Myb and Ets proteins, either alone or in combination, were constructed. We found that in the myeloid lineage v-myb, but not v-ets, induces cell transformation. In the erythroid lineage both v-myb and v-ets weakly transform erythroblast-like cells. These cells exhibit a mature phenotype and a low self-renewal capacity. The transforming efficiency of the two oncogenes is enhanced if they are coexpressed as separate proteins or as a fusion protein, the transformed cells displaying an increased self-renewal capacity. Interestingly, however, cells transformed by the Myb-Ets fusion protein have a distinct phenotype in that they are very immature. These results demonstrate that v-myb and v-ets can cooperate in the transformation of erythroid cells both in trans and in cis and that the mode of cooperation is reflected by the differentiation phenotypes of the transformed cells.

Elk , Tissue-Specific ets -Related Genes on Chromosomes X and 14 Near Translocation Breakpoints

The myb-ets-containing acute leukemia virus, E26, transforms myeloblasts and erythroblasts in culture and causes a mixed erythroid and myeloid leukemia in chicks. Genes (ets-1, ets-2, and erg) with variable relatedness to the v-ets oncogene of the E26 virus have been identified, cloned, and characterized in several species. Two new members (elk-1 and elk-2) of the ets oncogene superfamily have now been identified. Nucleotide sequence analysis of the elk-1 cDNA clone revealed that this gene encodes a 428-residue protein whose predicted amino acid sequence showed 82% similarity to the 3' region of v-ets. The elk or related sequences appear to be transcriptionally active in testis and lung. The elk cDNA probe detects two loci in the human genome, elk-1 and elk-2, which map to chromosome regions Xp11.2 and 14q32.3, respectively. These loci are near the translocation breakpoint seen in the t(X;18) (p11.2;q11.2), which is characteristic of synovial sarcoma, and the chromosome 14q32 breakpoints seen in ataxia telangiectasia and other T cell malignancies. This suggests the possibility that rearrangements of elk loci may be involved in pathogenesis of certain tumors.

Properties of a murine retroviral recombinant of avian acute leukemia virus E26: a murine fibroblast assay for v-ets function

A replication-defective murine retroviral construct, termed pME26, was generated by inserting avian gag-myb-ets sequences derived from the cloned avian acute leukemia virus E26 into an Abelson murine leukemia virus-derived retroviral vector. ME26 virus can be rescued efficiently from transfected NIH 3T3 cells by replicating murine leukemia viruses. Either pME26-transfected nonproducers or ME26 virus-infected NIH 3T3 cells expressed a 135-kilodalton fusion protein (p135) which was detectable by immunoprecipitation with antiserum directed against avian leukemia virus p27gag, myb or ets oncogene protein, or murine leukemia virus p15gag and was principally localized in the nucleus. NIH 3T3 cells infected with ME26 exhibited morphological alterations and increased proliferation in reduced serum and formed small colonies in agar suspension. Discrete foci could be readily recognized in cells maintained in a defined medium containing 0.03 to 0.1% calf serum. In newborn NFS/N mice, ME26 induced a significantly higher mortality and incidence of erythroid and myeloid leukemias. Analysis of a series of mutants affecting the expression of various portions of p135 indicated that the v-ets gene acts to mitogenically stimulate the proliferation of NIH 3T3 fibroblasts and reduces or abolishes their serum dependence. These properties provide an assay system to study functions of the ets gene family.

Transformation of avian lymphoid cells by reticuloendotheliosis virus.

Avian reticuloendotheliosis virus (REV-T) is the most virulent of all retroviruses, inducing an invariably fatal leukemia in chickens with a latent period of 7-10 days. Unlike avian cells transformed by other acutely transforming viruses, lymphoid cells transformed by REV-T are immortalized. Furthermore, in vitro derived, REV-T transformed cells which do not produce virus are tumorigenic and induce lethal reticuloendotheliosis when injected into histocompatible birds. Thus REV-T transforms its target cell both in vitro and in vivo. In addition this transformation is independent of any helper virus functions. Like other acute leukemia viruses, REV-T is replication-defective and must co-replicate with a reticuloendotheliosis associated virus (REV-A). During evolution, a substantial portion of its genome has been deleted and replaced with a host-derived genetic sequence, designated v-rel. Presumably, the v-rel oncogene was transduced from a normal turkey DNA locus, c-rel. There are 9 regions of homology between c-rel and v-rel, however, several differences exist between these genes, suggesting that transformation by REV-T results from the production of an altered v-rel protein. The v-rel sequence is distinct from other known oncogenes and encodes a 57-kDa phosphoprotein. In REV-T transformed cells, this pp57v-rel protein is localized in the cytoplasm. The product of the v-rel oncogene is present at a low level, representing only about 0.003% of total methionine-labelled protein. In addition, pp57v-rel is relatively stable, having an estimated half-life of 4-10 h. The v-rel protein when purified close to homogeneity is complexed with a 40-kDa cellular phosphoprotein in transformed lymphoid cells and possesses serine kinase activity. This review discusses the molecular aspects of transformation by REV-T in the context of other oncogene-encoded proteins.

A new antigen identified by the monoclonal antibody UCHB 1 delivers a costimulatory signal to a subset of human B cells.

A novel anti-B cell monoclonal antibody UCHB1 is described which detects an antigen present on a selected subpopulation of both normal and leukemic B cells. Approximately 20% of normal tonsil and peripheral blood (PB) B cells are UCHB 1+ and the majority of B cells from all cases of prolymphocytic leukemia (PLL) tested, together with a low, variable percentage of PB B cells from non-Hodgkin's lymphoma patients with centroblastic centrocytic leukemia also express this antigen. Chronic lymphocytic leukemia and hairy cell leukemia B cells, pre-B acute lymphoblastic leukemia and Epstein-Barr virus transformed cell lines all lack UCHB 1 positivity as do all non-B lineage leukocytes and cell lines so far tested. In tonsil sections UCHB 1 staining is almost completely confined to surface IgM+ mantle zone lymphocytes and follicular dendritic cells, but not B cells, within the germinal centers. UCHB 1 can induce a rise in the level of intracellular free calcium ([Ca2+]i) in PLL B cells and low concentrations of monoclonal antibody can result in the entry of these cells into cell cycle in the absence of additional factors. The proliferative effect of UCHB 1 is greatly enhanced in the presence of Staphylococcus aureus Cowan and, to a lesser extent, phorbol ester. A similar costimulatory effect is exerted on a proportion of normal tonsil B cells although here, despite inducing a rise in [Ca2+]i, UCHB 1 alone does not cause cells to proliferate. UCHB 1 may well prove to be a useful antibody both to distinguish between different B cell leukemias and to study the phenotype and function of leukemic and normal B cell subpopulations.

Erg, a human ets-related gene on chromosome 21: alternative splicing, polyadenylation, and translation.

The avian acute leukemia virus E26 induces a mixed erythroid-myeloid leukemia in chickens and carries two distinct oncogenes, v-myb and v-ets. Recently, a novel gene named erg, closely related to the v-ets oncogene, was identified in human COLO 320 cells and the nucleotide sequence of its approximately 5.0-kilobase transcript, erg 1 was determined. In the present study, the nucleotide sequence of the alternatively spliced transcript, erg 2, was found to differ from erg 1 by a splicing event that causes a coding frameshift near the amino terminus, resulting in an additional 99-amino acid insertion at the amino-terminus. Expression of complementary DNAs for the two transcripts in vitro resulted in synthesis of polypeptides of approximately 41 and 52 kilodaltons, suggesting the use of alternative translation initiation codons in the case of erg proteins. The erg gene was localized by somatic cell genetic analysis to human chromosome 21. It is proposed that alternative sites of splicing and polyadenylation, together with alternative sites of translation initiation, allow the synthesis of two related polypeptides from a single erg gene transcriptional unit.

Regulation of interferon receptor expression in human blood lymphocytes in vitro and during interferon therapy.

Interferons (IFN) elicit antiviral and antineoplastic activities by binding to specific receptors on the cell surface. The binding characteristics of IFN to human lymphocytes were studied using IFN alpha 2 labeled with 125I to high specific activity. The specific binding curves generated were analyzed by the LIGAND program of Munson and Rodbard to determine receptor numbers. The number of receptors in peripheral blood lymphocytes (PBL) and tonsillar B-lymphocytes (TBL) from normal individuals were 505 +/- 293 (n = 10) and 393 +/- 147 (n = 3) respectively. When these cells were preincubated in vitro with unlabeled IFN alpha 2, the receptor number decreased to 82 +/- 45 and 61 +/- 16 respectively. Receptor binding activities recovered gradually over a period of 72 h when the cells were incubated in IFN-free medium. This recovery of receptors could be blocked by the addition of actinomycin D to the incubation medium. A similar decrease in receptor expression was observed in vivo in PBL from patients being treated daily with 5 X 10(6) units/m2 per d of IFN alpha 2 by subcutaneous injection, for acute lymphoblastic leukemia or papilloma virus infections. Receptor numbers in PBL in vivo were further reduced concurrent with the progression of IFN therapy. Thus the reduction in IFN receptor expression observed in vitro can be demonstrated in vivo. These studies indicate that monitoring IFN receptor expression in vivo can provide information regarding the availability of IFN receptors at the cell surface for the mediation of IFN actions during the course of IFN therapy.

Identification and preferential expression in thymic and bursal lymphocytes of a c-ets oncogene-encoded Mr 54,000 cytoplasmic protein.

The avian retrovirus E26 is unique among acute leukemia viruses in its ability to induce transformation of cells belonging to either the myeloid or erythroid lineage. The genome of E26 carries two oncogenes, v-myb and v-ets, that are derived from distinct cellular loci, c-myb and c-ets. We have constructed a plasmid vector that allows expression of part of the coding region of v-ets in a bacterial host. Antisera to the bacterially synthesized ets protein specifically precipitated the E26-encoded P135gag-myb-ets transforming protein. These antisera permitted us to identify a chicken c-ets-encoded protein of Mr 54,000 (P54c-ets) that shares 7 out of 10 of its major [35S]methionine-containing tryptic peptides with the v-ets-encoded domain of P135gag-myb-ets. Unlike P135gag-myb-ets and the Mr 75,000 translation product of c-myb (P75c-myb), which are nuclear proteins, P54c-ets was found to be predominantly cytoplasmic. P54c-ets is expressed at low levels in most cell lines and tissues tested, including bone marrow cells and circulating lymphocytes. P54c-ets, together with a minor but closely related Mr 56,000 protein, was found to be expressed at high levels in chicken thymocytes and bursal lymphocytes.

Oncogenes in Retroviruses and Cells: Biochemistry and Molecular Genetics

This chapter discusses the biochemistry and the molecular genetics of a particular set of oncogenes, originally identified as transforming genes of avian acute leukemia viruses. This group of retroviruses played a key role in establishing the concept that normal cells contain multiple genes with the potential to become dominant oncogenic determinants. Six different cell-derived oncogenes were identified in this virus group alone, myc oncogene among them— by now one of the most intensively studied genes implicated in viral and nonviral carcinogenesis. In addition, several basic features and some unique variations of oncogene structure and expression were first diagnosed in this virus group. Also, a crucial progress in the search for physiological functions of cellular alleles of oncogenes was achieved by the discovery of close sequence homology between an oncogene of an avian acute leukemia virus and a human gene encoding a growth factor receptor, Hence, the oncogenes of avian acute leukemia viruses serve in the chapter as a model case to review the current knowledge about the structure and function of eukaryotic genes involved in malignant cell transformation.

Molecular analysis of myc gene mutants.

The myc gene was first Identified as a specific cellular sequence In the avian acute leukemia virus MC29 (Mellon et al., 1978; Roussel et al., 1979). Analysis of this virus has led to an extensive characterization of this sequence, the cellular homologue from which It was derived, and the gene product encoded by the virus (Hayman, 1983). The myc gene as found In MC29 Is closely related to the cellular sequence, c-myc (Watson et al., 1983). Only nine nucleotide changes are seen between MC29 v-myc and c-myc giving rise to seven amino acid changes. In addition v-myc contains ten base pairs of coding sequence at the 5’ end not found In c-myc The c-myc gene has three exons (two of which are coding exons) and two introns.KeywordsTarget Cell SpecificityDeletion SizePathogenic SpectrumDifferentiation State TumorMC29 proteInThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The proto-oncogene c-ets is preferentially expressed in lymphoid cells.

The transforming sequences of the avian acute leukemia virus, E26, contain two distinct oncogenes, v-mybE and v-ets, fused together. By using a probe containing v-ets sequences, polyadenylated transcripts of the c-ets proto-oncogene were detected in avian tissues; they included a major 7.0-kilobase and a minor 2.0-kilobase species. These c-ets mRNAs were detected at high levels only in lymphoid organs and in avian T and B lymphoid cell lines. A similar pattern of c-ets transcription was observed in human hematopoietic cell lines, with transcripts detected in lymphoid B and T cells but not in erythroid or myeloid cells. The E26 oncogene was inserted into an inducible expression vector, and a 90-kilodalton protein (bp90) was produced in bacteria. Rabbit antisera raised to purified bp90 precipitated P135gag-mybE-ets, the v-mybE-ets polyprotein expressed in E26-transformed cells, and also reacted with p50v-mybA, the transforming protein of the avian myeloblastosis virus. Antiserum to bp90 was absorbed with a bacterially synthesized v-mybA protein to remove anti-myb activity. The absorbed anti-bp90 serum retained the ability to immunoprecipitate P135gag-mybE-ets from E26-transformed cells and specifically reacted with a 56-kilodalton polypeptide (p56) detected in chicken lymphoid organs and in T and B lymphocytes of both avian and human origin. The data suggest that p56 is a translational product of the c-ets proto-oncogene and imply that p56 may be involved in regulating the growth of lymphoid cells.

The proto-oncogene c-ets is preferentially expressed in lymphoid cells.

The transforming sequences of the avian acute leukemia virus, E26, contain two distinct oncogenes, v-mybE and v-ets, fused together. By using a probe containing v-ets sequences, polyadenylated transcripts of the c-ets proto-oncogene were detected in avian tissues; they included a major 7.0-kilobase and a minor 2.0-kilobase species. These c-ets mRNAs were detected at high levels only in lymphoid organs and in avian T and B lymphoid cell lines. A similar pattern of c-ets transcription was observed in human hematopoietic cell lines, with transcripts detected in lymphoid B and T cells but not in erythroid or myeloid cells. The E26 oncogene was inserted into an inducible expression vector, and a 90-kilodalton protein (bp90) was produced in bacteria. Rabbit antisera raised to purified bp90 precipitated P135gag-mybE-ets, the v-mybE-ets polyprotein expressed in E26-transformed cells, and also reacted with p50v-mybA, the transforming protein of the avian myeloblastosis virus. Antiserum to bp90 was absorbed with a bacterially synthesized v-mybA protein to remove anti-myb activity. The absorbed anti-bp90 serum retained the ability to immunoprecipitate P135gag-mybE-ets from E26-transformed cells and specifically reacted with a 56-kilodalton polypeptide (p56) detected in chicken lymphoid organs and in T and B lymphocytes of both avian and human origin. The data suggest that p56 is a translational product of the c-ets proto-oncogene and imply that p56 may be involved in regulating the growth of lymphoid cells.

Molecular analysis of myc gene mutants.

We describe the generation and characterization of a series of deletion mutants of the avian acute leukaemia virus MC29 which allow the study of the function of the myc in transformation of quail embryo fibroblasts in vitro and tumour induction in vivo. These mutants, which are deleted in the 3' portion of the myc gene, fail to transform macrophages in vitro or induce tumours in vivo but are still able to transform morphologically fibroblasts. From one of these mutants a 'recovered' MC29 virus was generated which, like wild type MC29, transformed fibroblasts and macrophages in vitro. When tested in vivo this virus induced lymphomas of T and B cells rather that the endotheliomas induced by wild type MC29. This system allows us to investigate another question which is the mechanism by which the virus (or oncogene it contains) preferentially transforms one cell type.

Molecular cloning of proviral DNA and structural analysis of the transduced myc oncogene of avian oncovirus CMII.

Molecularly cloned proviral DNA of avian oncogenic retrovirus CMII was isolated by screening a genomic library of a CMII-transformed quail cell line with a myc-specific probe. On a 10.4-kilobase EcoRI fragment, the cloned DNA contained 4.4 kilobases of CMII proviral sequences extending from the 5' long terminal repeat to the EcoRI site within the partial (delta) complement of the env gene. The gene order of CMII proviral DNA is 5'-delta gag-v-myc-delta pol-delta env-3'. All three structural genes are partially deleted: the gag gene at the 3' end, the env gene at the 5' end, and the pol gene at both ends. The delta gag (0.83 kilobases)-v-myc (1.50 kilobases) sequences encode the p90gag-myc transforming protein of CMII. In comparison with the p110gag-myc protein of acute leukemia virus MC29, p90gag-myc lacks amino acids corresponding to additional 516 bases of gag sequences and 12 bases of 5' v-myc sequences present in the MC29 genome. Nucleotide sequence analysis of CMII proviral DNA at the delta gag-v-myc and the v-myc-delta pol junctions revealed significant homologies between avian retroviral structural genes and the cellular oncogene c-myc precisely at the positions corresponding to the gene junctions in CMII. Furthermore, the delta gag-v-myc junction in CMII corresponds to sequence elements in gag and C-myc that are possible splicing signals. The data suggest that transduction of cellular oncogenes may involve RNA splicing and recombination with homologous sequences on retroviral vectors. Different sequence elements of both the retroviral vectors and the c-myc gene recombined during genesis of highly oncogenic retroviruses CMII, MC29, or MH2.

Temperature-sensitive viral RNA expression in Moloney murine sarcoma virus ts110-infected cells.

We examined the mos-specific intracellular RNA species in 6m2 cells, an NRK cell line nonproductively infected with the ts110 mutant of Moloney murine sarcoma virus. These cells present a normal phenotype at 39 degrees C and a transformed phenotype at 28 or 33 degrees C, expressing two viral proteins, termed P85gag-mos and P58gag, at 28 to 33 degrees C, whereas only P58gag is expressed at 39 degrees C. It has been previously shown that 6m2 cells contain two virus-specific RNA species, a 4.0-kilobase (kb) RNA coding for P58gag and a 3.5-kb RNA coding for P85gag-mos. Using both Northern blot and S1 nuclease analyses, we show here that the 3.5-kb RNA is the predominant viral RNA species in 6m2 cells grown at 28 degrees C, whereas only the 4.0-kb RNA is detected at 39 degrees C. During temperature shift experiments, the 3.5-kb RNA species disappears after a shift from 28 to 39 degrees C and is detected again after a shift back from 39 to 28 degrees C. By Southern blot analysis, we have detected only one ts110 proviral DNA in the 6m2 genome. This observation, as well as previously published heteroduplex and S1 nuclease analyses which showed that the 3.5-kb RNA species lacks about 430 bases found at the gag gene-mos gene junction in the 4.0-kb RNA, suggests that the 3.5-kb RNA is a splicing product of the 4.0-kb RNA. The absence of the 3.5-kb RNA when 6m2 cells are grown at 39 degrees C indicates that the splicing reaction is thermosensitive. The splicing defect of the ts110 Moloney murine sarcoma virus viral RNA in 6m2 cells cannot be complemented by acute Moloney murine leukemia virus superinfection, since no 3.5-kb ts110 RNA was detected in acutely superinfected 6m2 cells maintained at 39 degrees C. The spliced Moloney murine leukemia virus env mRNA, however, is found in acutely infected cells maintained at 39 degrees C, suggesting that the lack of ts110 viral RNA splicing at 39 degrees C is not due to an obvious host defect. In sharp contrast, however, 6m2 cells chronically superinfected with Moloney murine leukemia virus produce a 3.5-kb RNA species at 39 degrees C as well as at 28 degrees C and contain proviral DNAs corresponding to the two viral RNA species.(ABSTRACT TRUNCATED AT 400 WORDS)